Compositions and methods relating to argininosucccinate synthetase

ABSTRACT

Processes and compositions for the therapeutic treatment of pathogenic Gram-negative bacterial infection are provided whereby arginino succinate synthetase or PEGylated arginino succinate synthetase is administered to a subject to inactivate endotoxin thereby reducing the likelihood of bacterial sepsis and improving patient outcome.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/298,309 filed Jan. 26, 2010, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods fordetection and treatment of microbial infection in a subject. In specificembodiments, the present invention relates to compositions and methodsfor detection and treatment of exposure to bacterial endotoxin in asubject.

BACKGROUND OF THE INVENTION

Infection by Gram-negative pathogens is associated with particularlysevere pathology, such as sepsis and endotoxic shock. Despite theadvances in understanding of pathophysiology of endotoxic shock andsepsis, therapies remain largely symptomatic and supportive.

Sepsis is generally characterized by multi-organ failure includingkidney, liver, heart and brain due to bacterial toxins absorbed frominfected wounds and circulating in blood. Due to the spread ofresistance against conventional antimicrobials in last decade, newagents and new compositions and methods are needed to counter septicchallenges. Methods and compositions for detection of bacterialendotoxin exposure in a subject are required for timely and appropriatetherapeutic intervention.

SUMMARY OF THE INVENTION

The following summary of the invention is provided to facilitate anunderstanding of some of the innovative features unique to the presentinvention and is not intended to be a full description. A fullappreciation of the various aspects of the invention can be gained bytaking the entire specification, claims, drawings, and abstract as awhole.

Processes of treating exposure of a subject to a bacterial endotoxin areprovided including administering a therapeutically effective amount ofargininosuccinate synthetase to a subject in need thereof.

An argininosuccinate synthetase as used in the inventions is optionallyrecombinantly expressed. In some embodiments, the argininosuccinatesynthetase has an amino acid sequence of SEQ ID NO: 1, or a variantthereof, optionally being a truncated human argininosuccinatesynthetase. Truncated argininosuccinate synthetase optionally retains anLPS binding site.

In some embodiments, argininosuccinate synthetase is PEGylated. APEGylated argininosuccinate synthetase is optionally PEGylated with aPEG comprising PEG₁₂. PEGylated argininosuccinate synthetase isoptionally PEGylated with an unbranched PEG, a branched PEG, orcombinations thereof. Several ratios of argininosuccinate synthetase toPEG are operable. In some embodiments the argininosuccinate synthetaseand PEG are present in a ratio ranging from 1:1 to 1:400, respectively.PEGylation of argininosuccinate synthetase is optionally random orsite-directed. Site-directed PEGylation is optionally directed toargininosuccinate synthetase lysines, histidines, cysteines, orcombinations thereof. Optionally, PEGylation includes at least onemolecule of PEG₁₂, optionally exclusively PEG₁₂. Optionally, PEGmolecules are associated with argininosuccinate synthetase by a covalentamide bond. PEG molecules attached to argininosuccinate are optionallylinear, branched, or a combination thereof.

A subject in the inventive processes is optionally a human. In someembodiments, a subject is infected with one or more Gram-negativebacteria such that the infection produces a subject in need of therapy.

Also provided are processes of detecting exposure to bacterial endotoxinby a subject that includes obtaining a sample from a subject exposed toa bacterial endotoxin prior to obtaining the sample, and determining thepresence, level, or activity of argininosuccinate synthetase in thesample from the subject. A sample is optionally blood or a fraction ofblood, optionally serum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates increases in endogenous argininosuccinate synthetasein serum from subjects challenged with, either LPS alone or along withthe liver injury priming agent D-galactosamine;

FIG. 2 are examples of rASS or PEGylated-rASS illustrated followingSDS-PAGE stained with either Coomassie blue or visualized by westernblotting;

FIG. 3 illustrates growth inhibition of E. coli by rASS orPEGylated-rASS;

FIG. 4 illustrates attenuation of LPS toxicity by rASS in macrophagecell cultures;

FIG. 5 illustrates decreases in LPS induced cellular cytotoxicity byrASS as measured by reduction in breakdown in cytoskeletal αII-spectrin(A) or in an MTS reduction assay (B);

FIG. 6 illustrates reduction in LPS-induced cytotoxicity in a rASSdose-dependent fashion;

FIG. 7 illustrates beneficial effects of rASS treatment on mouse (A) andrat (B) survival after challenge with LPS;

FIG. 8 illustrates suppression of TNF-α release (A) and C-reactiveprotein (B) levels by rASS in rat endotoxemia;

FIG. 9 illustrates inhibition of serum LDH release in subjects bysimultaneous or sequential administration of rASS;

FIG. 10 illustrates the nearly 3-fold higher activity ofPEGylated-rASS/mg protein vs. non-PEGylated rASS;

FIG. 11 illustrates that PEGylated-rASS is significantly more stable,and is retained in circulation for a much longer time after i.p.injection into subjects vs. non-PEGylated rASS.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following description of particular embodiment(s) of the inventionis merely exemplary in nature and is in no way intended to limit thescope of the invention, its application, or uses, which may, of course,vary. The invention is described with relation to the non-limitingdefinitions and terminology included herein. These definitions andterminology are not designed to function as a limitation on the scope orpractice of the invention but are presented for illustrative anddescriptive purposes only.

Compositions and methods are provided for detection and treatment ofbacterial endotoxin exposure in a subject. As such, the invention hasutility for detecting the presence of endotoxin in a sample or anorganism and for the treatment of infection by Gram-negative bacteria.

The terms “bacterial endotoxin,” “lipopolysaccharide” and “LPS” are usedinterchangeably herein to refer to this well-known structural componentof the outer membrane of Gram-negative bacteria that is referred tointerchangeably in the art.

Exposure of a subject to bacterial endotoxin occurs most commonly whenthe subject is infected by Gram-negative bacteria. Illustrative examplesof Gram-negative bacteria illustratively include Escherichia coli,Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae,Bordetella pertussis and Vibrio cholerae.

Methods of treating effects of exposure to bacterial endotoxin areprovided according to embodiments of the present invention whichincludes administering argininosuccinate synthetase, or alternativelyknown as argininosuccinate synthase (ASS) to a subject having, suspectedof having, or at risk for, infection by endotoxin-containing bacteriaand/or exposure to material suspected of containing bacterial endotoxin.

The terms “argininosuccinate synthetase” and “ASS” are usedinterchangeably herein to refer to the enzyme argininosuccinatesynthetase, optionally human derived, optionally identified herein asSEQ ID NO: 1, or variants thereof.

In addition to the argininosuccinate synthetase protein of SEQ ID NO: 1,the term argininosuccinate synthetase” encompasses variants of SEQ IDNO.1 which may be included in compositions and methods of the presentinvention. As used herein, the term “variant” refers to naturallyoccurring genetic variations of the SEQ ID NO.1 and recombinantlyprepared variations of SEQ ID NO.1, each of which contain one or morechanges in its amino acid sequence compared to SEQ ID NO.1. Such changesinclude those in which one or more amino acid residues have beenmodified by amino acid substitution, addition, or deletion. The term“variant” encompasses orthologs of human argininosuccinate synthetase,including, for example, mammalian and bird argininosuccinate synthetase,particularly argininosuccinate synthetase orthologs from non-humanprimates, cats, dogs, cows, horses, rodents, pigs, sheep, goats, fishand poultry.

Variants are argininosuccinate synthase optionally have at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO: 1.

Mutations can be introduced using standard molecular biology techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis. One ofskill in the art will recognize that one or more amino acid mutationscan be introduced without altering the functional properties of theargininosuccinate synthetase protein of SEQ ID NO: 1. For example, oneor more amino acid substitutions, additions, or deletions can be madewithout altering the functional properties of the argininosuccinatesynthetase protein of SEQ ID NO: 1.

Conservative amino acid substitutions can be made in argininosuccinatesynthetase protein of SEQ ID NO: 1 to produce argininosuccinatesynthetase protein variants. Conservative amino acid substitutions areart recognized substitutions of one amino acid for another amino acidhaving similar characteristics. For example, each amino acid may bedescribed as having one or more of the following characteristics:electropositive, electronegative, aliphatic, aromatic, polar,hydrophobic and hydrophilic. A conservative substitution is asubstitution of one amino acid having a specified structural orfunctional characteristic for another amino acid having the samecharacteristic. Acidic amino acids include aspartate, glutamate; basicamino acids include histidine, lysine, arginine; aliphatic amino acidsinclude isoleucine, leucine and valine; aromatic amino acids includephenylalanine, glycine, tyrosine and tryptophan; polar amino acidsinclude aspartate, glutamate, histidine, lysine, asparagine, glutamine,arginine, serine, threonine and tyrosine; and hydrophobic amino acidsinclude alanine, cysteine, phenylalanine, glycine, isoleucine, leucine,methionine, proline, valine and tryptophan; and conservativesubstitutions include substitution among amino acids within each group.Amino acids may also be described in terms of relative size, alanine,cysteine, aspartate, glycine, asparagine, proline, threonine, serine,valine, all typically considered to be small.

Argininosuccinate synthetase variants can include synthetic amino acidanalogs, amino acid derivatives and/or non-standard amino acids,illustratively including, without limitation, alpha-aminobutyric acid,citrulline, canavanine, cyanoalanine, diaminobutyric acid,diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid,homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine,homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine,and ornithine.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:22642268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Suchan algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searchesare performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules of the present invention. BLAST proteinsearches are performed with the XBLAST program parameters set, e.g., toscore 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule of the present invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST are utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively,PSI BLAST is used to perform an iterated search which detects distantrelationships between molecules (Id.). When utilizing BLAST, GappedBLAST, and PSI Blast programs, the default parameters of the respectiveprograms (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBIwebsite). Another preferred, non limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, 1988, CABIOS 4:11 17. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 is used.

The percent identity between two sequences is determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

Argininosuccinate synthetase included in methods and compositions of thepresent invention is optionally produced using recombinant nucleic acidtechnology. Argininosuccinate synthetase production includes introducinga recombinant expression vector encompassing a DNA sequence encodingargininosuccinate synthetase.

A nucleic acid sequence encoding argininosuccinate synthetase introducedinto a host cell to produce argininosuccinate synthetase encodes SEQ IDNO: 1. In embodiments of the present invention, the nucleic acidsequence identified herein as SEQ ID NO: 2 encodes SEQ ID NO: 1 and isincluded in an expression vector and expressed to produceargininosuccinate synthetase.

It is appreciated that due to the degenerate nature of the genetic code,nucleic acid sequences substantially identical to SEQ ID NO: 2 encodeargininosuccinate synthetase and variants of argininosuccinatesynthetase, and that such alternate nucleic acids may be included in anexpression vector and expressed to produce argininosuccinate synthetaseand variants of argininosuccinate synthetase.

A nucleic acid sequence which is substantially identical to SEQ ID No. 2is characterized as having a complementary nucleic acid sequence capableof hybridizing to SEQ ID No. 2 under high stringency hybridizationconditions.

The term “nucleic acid” refers to RNA or DNA molecules having more thanone nucleotide in any form including single-stranded, double-stranded,oligonucleotide or polynucleotide. The term “nucleotide sequence” refersto the ordering of nucleotides in an oligonucleotide or polynucleotidein a single-stranded form of nucleic acid.

The term “complementary” refers to Watson-Crick base pairing betweennucleotides and specifically refers to nucleotides hydrogen bonded toone another with thymine or uracil residues linked to adenine residuesby two hydrogen bonds and cytosine and guanine residues linked by threehydrogen bonds. In general, a nucleic acid includes a nucleotidesequence described as having a “percent complementarity” to a specifiedsecond nucleotide sequence. For example, a nucleotide sequence may have80%, 90%, or 100% complementarity to a specified second nucleotidesequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of asequence are complementary to the specified second nucleotide sequence.For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementaryto the nucleotide sequence 5′-AGCT-3′. Further, the nucleotide sequence3′-TCGA- is 100% complementary to a region of the nucleotide sequence5′-TTAGCTGG-3′.

The terms “hybridization” and “hybridizes” refer to pairing and bindingof complementary nucleic acids. Hybridization occurs to varying extentsbetween two nucleic acids depending on factors such as the degree ofcomplementarity of the nucleic acids, the melting temperature, Tm, ofthe nucleic acids and the stringency of hybridization conditions, as iswell known in the art. The term “stringency of hybridization conditions”refers to conditions of temperature, ionic strength, and composition ofa hybridization medium with respect to particular common additives suchas formamide and Denhardt's solution. Determination of particularhybridization conditions relating to a specified nucleic acid is routineand is well known in the art, for instance, as described in J. Sambrookand D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press; 3rd Ed., 2001; and F. M. Ausubel, Ed., ShortProtocols in Molecular Biology, Current Protocols; 5th Ed., 2002. Highstringency hybridization conditions are those which only allowhybridization of substantially complementary nucleic acids. Typically,nucleic acids having about 85-100% complementarity are considered highlycomplementary and hybridize under high stringency conditions.Intermediate stringency conditions are exemplified by conditions underwhich nucleic acids having intermediate complementarity, about 50-84%complementarity, as well as those having a high degree ofcomplementarity, hybridize. In contrast, low stringency hybridizationconditions are those in which nucleic acids having a low degree ofcomplementarity hybridize.

The terms “specific hybridization” and “specifically hybridizes” referto hybridization of a particular nucleic acid to a target nucleic acidwithout substantial hybridization to nucleic acids other than the targetnucleic acid in a sample.

Stringency of hybridization and washing conditions depends on severalfactors, including the Tm of the probe and target and ionic strength ofthe hybridization and wash conditions, as is well-known to the skilledartisan. Hybridization and conditions to achieve a desired hybridizationstringency are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001;and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology,Wiley, 2002.

An example of high stringency hybridization conditions is hybridizationof nucleic acids over about 100 nucleotides in length in a solutioncontaining 6×SSC, 5×Denhardt's solution, 30% formamide, and 100micrograms/ml denatured salmon sperm at 37° C. overnight followed bywashing in a solution of 0.1×SSC and 0.1% SDS at 60° C. for 15 minutes.SSC is 0.15M NaCl/0.015M Na citrate. Denhardt's solution is 0.02% bovineserum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone. Under highlystringent conditions, SEQ ID No. 2 will hybridize to the complement ofsubstantially identical targets and not to unrelated sequences.

The term “expression vector” refers to a recombinant vehicle forintroducing a nucleic acid encoding argininosuccinate synthetase into ahost cell where the nucleic acid is expressed to produceargininosuccinate synthetase. In particular embodiments, an expressionvector including SEQ ID NO: 2 or a substantially identical nucleic acidsequence is expressed to produce argininosuccinate synthetase in cellscontaining the expression vector.

In addition to one or more nucleic acids encoding argininosuccinatesynthetase, one or more nucleic acid sequences encoding additionalproteins can be included in an expression vector. For example, suchadditional proteins include non-argininosuccinate synthetase proteinssuch as reporters, including, but not limited to, beta-galactosidase,green fluorescent protein and antibiotic resistance reporters.

Expression vectors are known in the art and include plasmids andviruses, for example. An expression vector contains a nucleic acid thatincludes segment encoding a polypeptide of interest operably linked toone or more regulatory elements that provide for transcription of thesegment encoding the polypeptide of interest. Such regulatory elementsinclude, but are not limited to, promoters, terminators, enhancers,origins of replication and polyadenylation signals.

In particular embodiments, the recombinant expression vector encodes atleast argininosuccinate synthetase of SEQ ID NO: 1, a protein having atleast 95% identity to SEQ ID NO: 1, or a protein encoded by a nucleicacid sequence substantially identical to SEQ ID NO: 2.

Expression of argininosuccinate synthetase using a recombinantexpression vector is accomplished by introduction of the expressionvector into a eukaryotic or prokaryotic host cell expression system suchas an insect cell, mammalian cell, yeast cell, bacterial cell or anyother single or multicellular organism recognized in the art. Host cellsare optionally primary cells or immortalized derivative cells.Immortalized cells are those which can be maintained in-vitro for atleast 5 replication passages.

Host cells containing the recombinant expression vector are maintainedunder conditions wherein argininosuccinate synthetase is produced. Hostcells may be cultured and maintained using known cell culture techniquessuch as described in Celis, Julio, ed., 1994, Cell Biology LaboratoryHandbook, Academic Press, N.Y. Various culturing conditions for thesecells, including media formulations with regard to specific nutrients,oxygen, tension, carbon dioxide and reduced serum levels, can beselected and optimized by one of skill in the art.

Argininosuccinate synthetase optionally has an amino acid sequenceidentical to an argininosuccinate synthetase found in nature.Illustratively, argininosuccinate synthetase has the sequence of SEQ IDNO: 1. Some embodiments of the invention use an argininosuccinatesynthetase variant optionally with an amino acid sequence that differsfrom a sequence found in nature. Variants of argininosuccinatesynthetase are optionally created by site directed mutagenesis of a wildtype nucleic acid sequence encoding argininosuccinate synthetase.Illustratively, the sequence of SEQ ID NO: 2 is used as a source nucleicacid sequence for site-directed mutagenesis.

In some embodiments, an argininosuccinate synthetase variant is createdthat maintains the LPS binding site, but is altered elsewhere in themolecule. A variant is optionally a truncated form of argininosuccinatesynthetase. A truncated argininosuccinate synthetase optionally retainsthe wild-type LPS binding site, but is truncated elsewhere in themolecule. Optionally, a variant also, or independently, alters the LPSbinding site so as to increase or decrease the argininosuccinatesynthetase affinity for LPS. Methods of site-directed mutagenesis andscreening for affinity between molecules are well known in the art.

Argininosuccinate synthetase is optionally PEGylated. A protein isPEGylated when bound either covalently or otherwise to one or moremolecules of polyethylene glycol (PEG) either directly or through alinker. Covalent attachment of the inert, non-toxic, biodegradablepolymer PEG, to molecules has important applications in biotechnologyand medicine. PEGylation of biologically and pharmaceutically activeproteins typically improve pharmacokinetics, resulting in sustainedduration, improve safety (e.g. lower toxicity, immunogenicity andantigenicity), increase efficacy, decrease dosing frequency, improvedrug solubility and stability, reduce proteolysis, and facilitatecontrolled drug release (Roberts et al., 2002, Adv Drug Deliv Rev,54:459-76; Harris & Chess, 2003, Nat Rev Drug Discov, 2:214-221). Eachof these positive attributes of PEGylation of proteins is attributableto the alteration in pharmacokinetics of the protein. In fact, it isrecognized in the art that PEGylation reduces biological activity invitro. This reduction in activity is overcome in vivo by longertherapeutic lifetime. (See Jevsevar S, et al., Biotechnol. J., 2010;5:113-128.)

The inventors unexpectedly discovered that PEGylation ofargininosuccinate synthetase not only increases stability of theprotein, but also improves the enzymatic activity of argininosuccinatesynthetase. As such, some embodiments of the invention includeadministration of a PEGylated argininosuccinate synthetase which isshows unexpectedly superior efficacy in vivo and in vitro.

Argininosuccinate synthetase is optionally homogenously orheterogeneously PEGylated with PEG molecules ranging from 3 to 1000 PEGunits, optionally from 3 to 100 PEG units, optionally from 3 to 50 PEGunits, or any value or subrange therebetween. The total molecular weightof PEG molecules bound to argininosuccinate synthetase is optionallyfrom 300 Daltons to 50,000 Daltons or any range or subdivisiontherebetween.

PEG molecules used to PEGylate argininosuccinate synthetase areoptionally linear, branched, or a combination thereof. In someembodiments, only linear PEG molecules are used to PEGylateargininosuccinate synthetase. In some embodiments, only branched PEGmolecules are used to PEGylate argininosuccinate synthetase. Therelative ratios of unbranched to branched PEG molecules are appreciatedto be any range suitable for improving the stability of the resultingPEGylated argininosuccinate synthetase. Illustratively, the ratio ofunbranched to branched PEG ranges from 0.001:1 to 1:0.001, or any valueor range therebetween.

The extend of PEGylation on each individual argininosuccinate synthetasemolecule optionally varies. The ratio of argininosuccinate synthetase toPEG is optionally anywhere from 1:1 to 1:500, or any value or rangetherebetween. In some embodiments, the ratio of argininosuccinatesynthetase to PEG is from 1:1 to 1:100, optionally 1:1 to 1:10,optionally, 1:1 to 1:5. In some embodiments the ratio ofargininosuccinate synthetase to PEG is 1:10. These ranges illustratemolar ratios whereby one mole of argininosuccinate synthetase is boundto anywhere from 1 to 500 moles of PEG species.

Numerous PEG species are operable to PEGylate argininosuccinatesynthetase. Illustrative examples include PEG-chlorotriazine, PEGsuccinimidyl succinate (SS-PEG), succinimidyl carbonate PEG (SC-PEG),the N-Hydroxysuccinimide PEGs including methyl succinimidyl PEG (MS-PEG)and trimethyl succinimidyl PEG (TMS-PEG), as well as other PEG moleculesknown in the art. Specific examples of PEG molecules operable hereininclude MS-PEG₁₂ and TMS-PEG₁₂.

PEGylation of argininosuccinate synthetase is illustratively random orsite-specific. Random PEGylation is illustratively achieved by a linkerthat interacts with proteins to form an amide linkage or a urethanelinkage. Amide linkages allow stable PEG binding to lysine residues aswell as the protein N-terminus. Urethane linkages are capable ofinteracting with lysine and histidine residues. A urethane linkage maybe relatively unstable compared to an amide linkage, but may have theadvantage in the preparation of pro-drug or controlled-releaseformulations of PEGylated argininosuccinate synthetase.

PEGylation of argininosuccinate synthetase is optionally site-specificmeaning that the preparation of PEGylated argininosuccinate synthetasetypically has increased uniformity over random PEGylation. PEGylation isoptionally specific to the N-terminus or other location onargininosuccinate synthetase or a variant thereof. Techniques forN-terminal and cysteine-specific PEGylation are well known in the art.N-terminal PEGylation is illustratively achieved with a PEG-aldehydereagent. PEGylation of thiol groups are illustratively achieved usingthiol specific reagents such as maleimide, pyridyl disulfide, and vinylsulfone, among others known in the art. Illustrative examples oftechniques suitable for site-directed PEGylation include transientdenaturing conditions of Veronese, F M, et al., Bioconjug. Chem., 2007;18:1824-1830. Alternatively, the methods presented in U.S. PatentApplication Publication Nos: 2010/0247508 or 2009/047500 may be used. Insome embodiments, free cysteines (or other specific amino acid, or aminoacid chemistries) are incorporated into the sequence ofargininosuccinate synthetase such as by site directed mutagenesis so asto create PEGylation sites. Another optional method for site-directedPEGylation is that of enzymatic PEGylation. Enzymatic PEGylation ofPEG-alkylamine reagents is illustratively described by Sato, H, Adv.Drug Deliv. Rev., 2002; 54:487-504.

The extent of PEGylation on argininosuccinate synthetase is optionallyhomogenous or heterogeneous throughout a therapeutically effectivesolution of PEGylated argininosuccinate synthetase. In some embodiments,more or either the linear or branched PEG is present in a sample. Assuch a solution of argininosuccinate synthetase optionally includesargininosuccinate synthetase molecules with uniform or varyingPEGylation type, extent, or mass.

Methods of preventing or treating a disease or disorder characterized bysigns and/or symptoms of exposure to bacterial endotoxin are providedaccording to the present invention which includes administering atherapeutically effective amount of a composition includingargininosuccinate synthetase to a subject in need thereof. In particularembodiments, a composition according to the present invention isadministered to a subject having a disease or disorder or at risk for adisease or disorder characterized by exposure of the subject tobacterial endotoxin.

Broadly described, a method according to embodiments of the presentinvention includes administration of argininosuccinate synthetase to anorganism, a cell or tissue, in vitro or in vivo.

Administration of argininosuccinate synthetase is optionally followed byassay of the effects of argininosuccinate synthetase in the subjectorganism, cell or tissue.

The term “therapeutically effective amount” as used herein is intendedto mean an amount of an inventive composition which is effective toalleviate, ameliorate or prevent a symptom or sign of a condition to betreated. In particular embodiments, a therapeutically effective amountis an amount which has a beneficial effect in a subject having signsand/or symptoms of exposure to bacterial endotoxin.

Thus, for example, in particular embodiments, treatment of a subject toprevent or treat effects of exposure to bacterial endotoxin in thesubject is characterized by prevention or amelioration of pathogeniceffects of bacterial endotoxin. Amelioration of signs and symptoms ofsepsis and/or endotoxic shock is assessed by techniques known in the artand described herein.

Signs and symptoms of exposure to bacterial endotoxin include, but arenot limited to, fever, rapid heartbeat, rapid respiration, low bloodpressure, local or generalized Shwartzman reaction and organ failure.

The term “subject” refers to any individual to whom a composition of thepresent invention is administered. The term “subject” includes mammalsand birds, particularly humans, non-human primates, cats, dogs, cows,horses, rodents, pigs, sheep, goats and poultry. A subject in need is asubject suffering from a form of bacterial sepsis. A subject in needoptionally suffers or suffered: an intravenous puncture; perforated,compromised, or ruptured intra-abdominal or pelvic structure;bacteruria; or other infection illustratively with the at least one ofthe pathogenic bacteria Streptococcus pneumonia, Neisseria meningitides,Staphylococcus aureus, Hemophilus influenzae, Klebsiella pneumonia,Legionella spp., Streptococcus agalactiae, E. coli, Klebsiellapneumoniae, Listeria monocytogenes, Enterococcus spp., Streptococcuspyogenes, Erysipelothrix rhusiopathiae, Aeromonas hydrophila, Vibriovulnificus, Clostridium perfringens, Salmonella spp., or otherpathogenic Gram-negative bacteria known in the art. When infection is bya bacteria normally found in a subject, a subject in need is a subjectsuffering a bacterial infection in a compartment other than that inwhich the bacteria is normally found. As such, the term “infected with”means the presence of a Gram-negative bacteria in a biologicalcompartment where the bacteria is not normally found.

The amount of a composition of the present invention administered to asubject and the route of administration depends on factors such as theseverity of an infection affecting the subject, the activity and rate ofexcretion of the argininosuccinate synthetase, and the general physicalcharacteristics of the subject including age, gender and body weight.One of skill in the art could determine a therapeutically effectiveamount and route of administration in view of these and otherconsiderations typical in medical practice.

Amounts of argininosuccinate synthetase used in a method to inhibitbacterial endotoxin will be determined by one of skill in the artwithout undue experimentation.

In general, a therapeutically effective amount of argininosuccinatesynthetase in a composition is in the range of about 0.001 mg/kg-100mg/kg body weight. In particular embodiments, a therapeuticallyeffective amount of argininosuccinate synthetase in a composition is inthe range of about 0.01-10 mg/kg, and in further embodiments, atherapeutically effective amount of argininosuccinate synthetase in acomposition is in the range of about 0.1-5 mg/kg. A therapeuticallyeffective amount of a composition of the present invention may bemanufactured and/or administered in single or multiple unit dose forms.

In some embodiments, a method according to the present inventionincludes administering a therapeutic agent in addition toargininosuccinate synthetase to a subject. A therapeutic agent may beany of various agents suitable for use in conjunction with ameliorationof infection by endotoxin-containing bacteria or exposure to bacterialendotoxin. For example, a therapeutic agent is an antibiotic in oneembodiment of the present invention. Antibiotics include, for example,aminoglycosides, amoxicillin, amphenicols, ansamycins, antibioticpolypeptides, beta-lactams, carbapenems, cephalosporins, cephamycins,oxacephems, lincosamides, macrolides, monobactams, nitrofurans,quinolones, sulfonamides, sulfones and tetracyclines.

In particular embodiments, a composition is provided according to thepresent invention which includes argininosuccinate synthetase and apharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” as used herein refers toa carrier or diluent that is generally non-toxic to an intendedrecipient and which does not significantly inhibit activity ofargininosuccinate synthetase or other active agent included in thecomposition.

A composition according to the present invention generally includesabout 0.1-99% of argininosuccinate synthetase.

Argininosuccinate synthetase is included in a composition of the presentinvention in the form of a free acid or free base in particularembodiments. In further embodiments, argininosuccinate synthetase isincluded in a composition in the form of a pharmaceutically acceptablesalt such as an acid or base addition salt. A pharmaceuticallyacceptable salt refers to any salt form of argininosuccinate synthetasethat is generally non-toxic to an intended recipient and which does notsignificantly inhibit activity of the argininosuccinate synthetase orother active agent included in the composition. Argininosuccinatesynthetase is included in a composition in the form of a hydrate inembodiments of the present invention.

An argininosuccinate synthetase prodrug is included in a compositionaccording to particular embodiments of the present invention. Anargininosuccinate synthetase prodrug is a form of argininosuccinatesynthetase covalently bound to a moiety which is released fromargininosuccinate synthetase yielding the intact activeargininosuccinate synthetase. Prodrug forms are well known in the art asexemplified in Sloan, K. B., Prodrugs, M. Dekker, New York, 1992; andTesta, B. and Mayer, J. M., Hydrolysis in drug and prodrug metabolism:chemistry, biochemistry, and enzymology, Wiley-VCH, Zurich, 2003.

More than one form of argininosuccinate synthetase is included in acomposition according to embodiments of the present invention. Thus, forexample, in particular embodiments human argininosuccinate synthetaseand one or more variants of human argininosuccinate synthetase are bothincluded in a composition.

Argininosuccinate synthetase is administered to a subject as an isolatedargininosuccinate synthetase protein according to embodiments of thepresent invention. The term “isolated argininosuccinate synthetaseprotein” indicates that the argininosuccinate synthetase protein hasbeen separated from biological materials, such as cells, cellular debrisand other proteins, which may be present in the system in which theargininosuccinate synthetase protein was produced. The term “isolated”argininosuccinate synthetase protein may, but does not necessarily,indicate that the argininosuccinate synthetase protein is purified.Purified argininosuccinate synthetase protein included in methods andcompositions of the present invention contains least about 1-100% of themass, by weight, such as about 25%, 50%, 75%, 85%, 95%, 99% or greaterthan about 99% of the mass, by weight, of the protein included.

In some embodiments, an expression vector including a nucleic acidencoding argininosuccinate synthetase is administered to a subject toproduce the argininosuccinate synthetase protein in vivo.

A composition according to the present invention may be formulated invarious forms. A composition formulated for oral administration may be asolid, semi-solid or liquid formulation prepared according to methodsknown in the art and including any of various conventionalpharmaceutical ingredients.

Numerous delivery systems are known and can be used to deliverargininosuccinate synthetase to a subject, illustratively includingliposomes and nanoparticles such as nanospheres, nanodendrimers,nanocolloids, nanodots, nanocolumns, and combinations of these. Furtherdescription of liposomes and methods relating to their preparation anduse may be found in Liposomes: A Practical Approach (The PracticalApproach Series, 264), V. P. Torchilin and V. Weissig (Eds.), OxfordUniversity Press; 2nd ed., 2003. Further aspects of nanoparticles aredescribed in S. M. Moghimi et al., FASEB J. 2005, 19, 311-30.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms,argininosuccinate synthetase is admixed with at least onepharmaceutically acceptable carrier such as a filler or extender, as forexample, starches, lactose, sucrose, glucose, mannitol, and silicicacid; a binder, as for example, carboxymethylcellulose, alignates,gelatin, polyvinylpyrrolidone, sucrose, and acacia; a humectant, as forexample, glycerol; a disintegrating agent, as for example, agar-agar,calcium carbonate, plant starches such as potato or tapioca starch,alginic acid, certain complex silicates, and sodium carbonate; asolution retarder, as for example, paraffin; an absorption accelerator,as for example, quaternary ammonium compounds; a wetting agent, as forexample, cetyl alcohol, glycerol monostearate, and glycols; anadsorbent, as for example, kaolin and bentonite; a buffering agent, suchas sodium citrate and dicalcium phosphate; and a lubricant, as forexample, talc, calcium stearate, magnesium stearate, solid polyethyleneglycols and sodium lauryl sulfate. Mixtures of these or otherpharmaceutically acceptable carriers may also be included in embodimentsof a composition of the present invention.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

The enteric coating is typically a polymeric material. Preferred entericcoating materials have the characteristics of being bioerodible,gradually hydrolyzable and/or gradually water-soluble polymers. Theamount of coating material applied to a solid dosage generally dictatesthe time interval between ingestion and drug release. A coating isapplied with to a thickness such that the entire coating does notdissolve in the gastrointestinal fluids at pH below 3 associated withstomach acids, yet dissolves above pH 3 in the small intestineenvironment. It is expected that any anionic polymer exhibiting apH-dependent solubility profile is readily used as an enteric coating inthe practice of the present invention to achieve delivery of the activeto the lower gastrointestinal tract. The selection of the specificenteric coating material depends on properties such as resistance todisintegration in the stomach; impermeability to gastric fluids andactive agent diffusion while in the stomach; ability to dissipate at thetarget intestine site; physical and chemical stability during storage;non-toxicity; and ease of application.

Suitable enteric coating materials illustratively include cellulosicpolymers such as hydroxypropyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose,cellulose acetate, cellulose acetate phthalate, cellulose acetatetrimellitate, hydroxypropylmethyl cellulose phthalate,hydroxypropylmethyl cellulose succinate and carboxymethylcellulosesodium; acrylic acid polymers and copolymers, preferably formed fromacrylic acid, methacrylic acid, methyl acrylate, ammoniummethylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl; vinylpolymers and copolymers such as polyvinyl pyrrolidone, polyvinylacetate, polyvinylacetate phthalate, vinylacetate crotonic acidcopolymer, and ethylene-vinyl acetate copolymers; shellac; andcombinations thereof. A particular enteric coating material is thoseacrylic acid polymers and copolymers available under the trade nameEUDRAGIT, Roehm Pharma (Germany). The EUDRAGIT series L, L-30D Scopolymers, and cross-linked polymers, see for example U.S. Pat. No.6,136,345, are suitable in particular applications since these areinsoluble in the stomach and dissolve in the intestine.

The enteric coating optionally contains a plasticizer to prevent theformation of pores and cracks that allow the penetration of the gastricfluids into the solid dosage. Suitable plasticizers illustrativelyinclude, triethyl citrate (Citroflex 2), triacetin (glyceryltriacetate), acetyl triethyl citrate (Citroflex A2), Carbowax 400(polyethylene glycol 400), diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, a coating composed of ananionic carboxylic acrylic polymer typically contains approximately 10%to 25% by weight of a plasticizer, particularly dibutyl phthalate,polyethylene glycol, triethyl citrate and triacetin. The coating canalso contain other coating excipients such as detackifiers, antifoamingagents, lubricants (e.g., magnesium stearate), and stabilizers (e.g.,hydroxypropylcellulose, acids and bases) to solubilize or disperse thecoating material, and to improve coating performance and the coatedproduct.

The enteric coating is applied to a solid dosage using conventionalcoating methods and equipment. For example, an enteric coating can beapplied to a solid dosage using a coating pan, an airless spraytechnique, fluidized bed coating equipment, or the like. Detailedinformation concerning materials, equipment and processes for preparingcoated dosage forms may be found in Pharmaceutical Dosage Forms:Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989),and in L. V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, 8th Ed. (Philadelphia, Pa.: Lippincott, Williams& Wilkins, 2004).

Liquid dosage forms for oral administration include a pharmaceuticallyacceptable carrier formulated as an emulsion, solution, suspension,syrup, or elixir in particular embodiments. In addition toargininosuccinate synthetase, the liquid dosage forms may contain one ormore pharmaceutically acceptable carriers commonly used in the art, suchas water or other solvents, solubilizing agents and emulsifiers, as forexample, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseedoil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil,glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acidesters of sorbitan or mixtures of these substances, and/or other suchconventional pharmaceutical ingredients.

A composition formulated for oral administration can also includeadjuvants, such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

Suspensions, in addition to argininosuccinate synthetase, may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitol esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar or tragacanth,or mixtures of these substances, and/or other such conventionalpharmaceutical ingredients.

In particular embodiments, a composition including argininosuccinatesynthetase of the present invention is formulated as a physiologicallyacceptable sterile aqueous or nonaqueous solution, dispersion,suspension, emulsion, or sterile powder for reconstitution into asterile injectable solution or dispersion. Examples of suitable aqueousand nonaqueous carriers, include diluents, solvents, and vehicles suchas water, ethanol, polyols such as propylene glycol, polyethyleneglycol, glycerol, and the like, and suitable mixtures thereof; vegetableoils such as olive oil; and injectable organic esters such asethyloleate. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants,such as sodium lauryl sulfate.

Such formulations are administered by a suitable route includingparenteral and oral administration. Administration may include systemicor local injection, such as intravenous injection.

A composition of the present invention may also contain one or moreadjuvants such as preserving, wetting, emulsifying, and dispensingagents. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol and sorbic acid. It may also be desirable toinclude an isotonic agent, exemplified by sugars and sodium chloride.Prolonged delivery of an injectable pharmaceutical form can be achievedby the use of agents delaying absorption, for example, aluminummonostearate and gelatin.

Detailed information concerning materials, equipment and processes forpreparing and manufacturing various dosage forms may be found inPharmaceutical Dosage Forms: Tablets, eds. H. A. Lieberman et al., NewYork: Marcel Dekker, Inc., 1989, and in L. V. Allen, Jr. et al., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed.,Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004. Furtherexamples and details of pharmacological formulations and ingredients arefound in standard references such as: A. R. Gennaro, Remington: TheScience and Practice of Pharmacy, Lippincott Williams & Wilkins, 20thed., 2003; L. V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Formsand Drug Delivery Systems, 8th Ed., Philadelphia, Pa., Lippincott,Williams & Wilkins, 2004; and J. G. Hardman et al., Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, McGraw-Hill Professional,10th ed., 2001.

A composition including argininosuccinate synthetase may be administeredby a systemic route and/or by a local route. Suitable routes ofadministration illustratively include intravenous, oral, buccal,parenteral, intrathecal, intracerebroventricular, intraperitoneal,ocular, intraocular, rectal, vaginal, subcutaneous, intradermal,intramuscular, topical, intranasal, otic and mucosal.

In further embodiments of inventive methods, argininosuccinatesynthetase presence, levels, and/or activity are assessed in a samplesuspected of exposure to bacterial endotoxin. It is a finding of thepresent invention that argininosuccinate synthetase levels and activityare elevated over normal levels and activities in samples obtained fromsubjects exposed to bacterial endotoxin.

Assays for argininosuccinate synthetase levels and/or activity areoptionally performed on any material suspected of having been exposed tobacterial endotoxin, such as a sample from a subject, cultured primarycells and/or tissues or cell lines. Assays for argininosuccinatesynthetase levels and/or activity are optionally performed using any ofvarious assay methods illustratively include enzyme-linked immunosorbentassay (ELISA), flow cytometry, immunoblot, immunoprecipitation,immunocytochemistry, radioimmunoassay, RT-PCR, northern blothybridization, dot blot hybridization, RNAase protection, or acombination of any of these. Assay methods may be used to obtainqualitative and/or quantitative results. Specific details of suitableassay methods for both qualitative and quantitative assay of a sampleare described in standard references, illustratively including E. Harlowand D. Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1988; F. Breitling and S. Diibel, RecombinantAntibodies, John Wiley & Sons, New York, 1999; H. Zola, MonoclonalAntibodies: Preparation and Use of Monoclonal Antibodies and EngineeredAntibody Derivatives, Basics: From Background to Bench, BIOS ScientificPublishers, 2000; B. K. C. Lo, Antibody Engineering: Methods andProtocols, Methods in Molecular Biology, Humana Press, 2003; F. M.Ausubel et al., Eds., Short Protocols in Molecular Biology, CurrentProtocols, Wiley, 2002; S. Klussman, Ed., The Aptamer Handbook:Functional Oligonucleotides and Their Applications, Wiley, 2006;Ormerod, M. G., Flow Cytometry: a practical approach, Oxford UniversityPress, 2000; Givan, A. L., Flow Cytometry: first principles, Wiley, NewYork, 2001; Gorczyca, W., Flow Cytometry in Neoplastic Hematology:morphologic-immunophenotypic correlation, Taylor & Francis, 2006; and J.Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 3rd Ed., 2001, or by methods describedherein.

A sample from a subject are illustratively a sample of a tissue, cells,a bodily fluid that may or may not include cells, or a sample obtainedfrom the environment such as soil, water, or other environmental sample.Illustrative examples of a sample include blood, plasma, serum, saliva,mucous, semen, tears, lymph, and urine. As such, obtaining a sample isby any method known in the art to acquire a sample illustrativelyincluding venipuncture to obtain whole blood whereby serum isillustratively achieved by clotting the blood and removing the solublefraction, plasma is obtained by centrifugation of whole blood andremoving the upper plasma section, or other standard collectiontechniques.

A method of treating exposure to bacterial endotoxin is applicable to ahuman subject as well as a non-human subject. In particular embodiments,a method of treating exposure to bacterial endotoxin includesadministration of argininosuccinate synthetase to a human, or non-humansubject such as non-human primates, cats, dogs, cows, horses, rodents,pigs, sheep, goats and poultry or other non-human mammal or bird.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES

Reagents

Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 and Salmonellaenterica typhimurium are purchased from Sigma (St. Louis, Mo., USA). Therat TNF-α and CRP ELISA kits are purchased from eBioscience (San Diego,Calif.), and BioVendor (Modrice, Czech Rep.) respectively. Antibodiesagainst polyhistidine, αII-spectrin and LPS core (clone WN1 222-5) arepurchased from Santa Cruz Biotechnology (Santa Cruz, Calif.), Biomol Co.(Plymouth Meeting, PA), and HyCult Biotechnology (Uden, Netherlands)respectively. Antibodies against human ASS are produced at BanyanBiomarkers, Inc. by standard techniques.

Example 1 LPS Treatment Induces Serum Accumulation of Endogenous ASSBiomarker in vivo

Adult male Sprague-Dawley rats (200-225 g) and Balb/c mice (19-22 g)(Harlan Laboratories Inc., Indianapolis, Ind.) are housed under constanttemperature (22° C.) and humidity with 12 hours light/dark cycle withaccess to chow and water ad libitum.

Levels of endogenous ASS in serum are determined after LPS treatment incombination with liver injury priming agent D-galactosamine (D-Gal).Lipopolysaccharide from E. coli (LPS, 10 μg/kg) plus D-galactosamine(D-Gal, 500 mg/kg), LPS alone (100 μg/kg), or saline are injectedintraperitoneally (i.p.) in Sprague-Dawley rats. Blood is collected fromheart of anesthetized animals 1 h, 2 h, 3 h, 24 h and 72 h after thetreatment, using at least 3 rats for each time point. As seen in FIG. 1,ASS is extremely sensitive marker of endotoxin-induced liver injury andsignificantly increased in serum within 1 hour following injection ofendotoxin and D-galactosamine (FIG. 1A). The ASS accumulation in bloodattains 1000 ng/ml in rats surviving 24 hours after treatment. In ratsrecovering from injection, ASS serum levels decline at 72 hours tonearly baseline, but are still elevated over control, saline-treatedrats (FIG. 1A). When LPS is injected at 10× fold higher dose withoutD-Gal, serum ASS increases exhibited a similar profile but the levelsare of significantly lower magnitude than in the presence ofD-galactosamine (FIG. 1B).

Example 2 Recombinant Argininosuccinate Synthetase Cloning, ProteinExpression and Purification

The coding region (residues 1-412) of the human argininosuccinatesynthetase gene (GenBank accession No. BC009243.2; Swiss-Prot nameASSY_HUMAN) is amplified using original clone from Open Biosystems(Huntsville, Ala.) (Clone ID: 3010137) as a template for PCRamplification. Primers are as follows: forward 32-mer (including ATG),5′-ATGTCCAGCAAAGGCTCCGTGGTTCTGGCCTA-3′(SEQ ID NO. 3); reverse 50-mer(created to contain a HindIII site and a C-terminal His-tag fusion),5′-TATAAAGCTTTCAATGGTGATGGTGATGATGTTTGGCAGTGACCTTGCTC-3′(SEQ ID NO 4).Thirty cycles of PCR are performed as the following: denaturation for 1min at 94° C., annealing for 1 min at 53° C., and elongation for 2 minat 72° C. This results in the amplification of a single product of thepredicted size for human ASS1-6×His fusion (1254 base pairs) that isgel-purified and directly ligated into pETBlue-1 vector using AccepTorVector kit (Novagen, Madison, Wis.). The correct sequence of the cDNA isverified by Sanger sequencing.

For the inducible expression of ASS1 cDNA under the control of the T7lacpromoter, the construct is transformed into the Tuner(DE3)pLacI E. colistrain (Novagen). After transformation by a standard heat shocktechnique and liquid-culture growth in Luria Broth (LB) using standardmethods, expression of recombinant human argininosuccinate synthetase(rASS) is induced by the addition of isopropyl-thiogalactopyranoside(IPTG). Induction conditions (16 hr at 18° C. in the presence of 0.5 mMIPTG) are optimized to achieve the highest yield of soluble rASS. Afterharvesting, the cell pellets are subjected to freezing at −70° C. andthawing at 37° C. and suspended in SoluLyse lysis buffer (GenLantis, SanDiego, Calif.) supplemented with protease inhibitor and DNAase. rASS ispurified using affinity chromatography on HisPur Cobalt spin column(Pierce, Rockford, Ill.). The binding/wash buffer contains 50 mM sodiumphosphate, 300 mM NaCl, and 10 mM imidazole pH 7.4. The elution bufferis identical except that the imidazole concentration is raised to 150mM. The protein is concentrated and the buffer exchanged forphosphate-buffered saline (PBS) using U-Tube concentrators (Novagen).The final buffer solution at pH 7.4 is supplemented with 1 mM citrullineand 1 mM aspartate to preserve protein solubility and catalyticactivity.

Affinity purified protein is ˜95% pure protein as indicated by CoomassieBlue staining (FIG. 2A).

PEGylated-rASS is prepared using either MS-PEG₁₂- or TMS-PEG₁₂ fromPierce Biotechnology, Rockford, Ill. Briefly, a solution of MS-PEG12- orTMS-PEG12 is prepared in anhydrous dimethylformamide and added dropwiseto a solution of rASS protein at ratios of 200:1 or 400:1, PEG to ASSrespectively. The reaction mixture is incubated at room temperature for0.5 h and the solution of protein is purified on a calibrated DesaltSpin Column by centrifugation at 1,000×g for 5 min. Purity andcompletion of reaction is evaluated using SDS-PAGE. (FIG. 2A, E)Immunogenic purity of rASS is established by western blot with antibodyagainst 6×His (FIG. 2B, E) as well as polyclonal antibody (ASS1) raisedin rabbits against an ASS-derived peptide (FIG. 2C, E).

Example 3 Bacterial Growth Kinetics

A strong physical interaction of ASS with LPS could be responsible for adeposition of de novo synthesized rASS into inclusion bodies in bacteriaat 37° C. The inhibitory effect of rASS or PEGylated-rASS on E. coli orB. subtilis bacterial growth is assessed as optical density at 600 nm atdifferent concentrations of rASS. E. coli (K-12 strain) expressing LPSon cell surface or B. subtilis (control) are allowed to grow in theabsence or presence of rASS or PEGylated-rASS (0.25 or 0.5 μg/ml) in a96-well plate in LB (Luria-Bertani) medium (Novagen) with a startingdensity of 0.06 optical units (at 600 nm). After each hour up to 6hours, OD at 600 nm is measured to determine bacterial growth rate. FIG.3 shows that 3 to 6 hours following addition to suspension cultures, 50μg/ml of rASS (FIG. 3A) or PEGylated rASS (FIG. 3C) significantlysuppresses E. coli growth demonstrating direct antibacterial effects ofthe rASS supplement. rASS and PEGylated-rASS similarly inhibit growth.rASS shows no apparent reduction in growth of B. subtilis. (FIG. 3B)

Example 4 Western Blot Analyses

For western blot analyses samples are homogenized on ice in western blotbuffer. The samples are subjected to SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and electroblotted onto polyvinylidenedifluoride membranes. Membranes are blocked in 10 mM Tris, pH 7.5, 100mM NaCl, and 0.1% Tween-20 containing 5% nonfat dry milk for 60 min atroom temperature. After overnight incubation with primary antibodies,proteins are detected either using secondary antibody conjugated tohorseradish peroxidase (HRP) and a chemiluminescence detection system,or secondary antibody conjugated to alkaline phosphatase (ALP) andcolorimetric detection system.

Example 5 Attenuation of LPS Toxicity by rASS in Macrophage CellCultures

The beneficial effects of rASS against LPS toxicity are examined onmammalian cells in culture using release of lactate dehydrogenase (LDH)into the medium as an indicator of LPS toxicity.

RAW 264.7 mouse macrophages are purchased from American Type CultureCollection (Manassas, Va.) and cultured in DMEM supplemented with 10%fetal bovine serum and antibiotic-antimycotic solution (100 U/mlpenicillin, 100 μg/ml streptomycin and 25 μg/ml amphotericin B) in 5%CO₂ at 37° C. Cultures are passaged every 3 to 5 days, and cells aredetached by brief trypsin treatment and visualized in an invertedmicroscope.

The macrophages are treated with LPS (0.1-1 μg/ml), rASS (1 and 10μg/ml) and anti-LPS core antibodies (0.1 μg/ml). At different timepoints cell-conditioned media from each well is saved for LDH releaseassay. Cells at the terminal time points (6 or 18 h) are washed andsubjected to MTS assay. The cells are lysed for western blot analyses ofall-spectrin breakdown products.

At 1 μg/ml, LPS from both E. coli, LPS(E) or S. enterica, LPS(S) exhibita remarkable cytotoxicity as indicated by a sharp increase of medium LDHlevels at 6 and 18 hours following LPS challenge (FIGS. 4 A and 4B).Pre-incubation of LPS with rASS (1 μg/ml) for 1 hour before additionsignificantly reduces the LDH release by two types of LPS at 6 and 18hours after treatment (FIGS. 4 A and 4B). In contrast, pre-incubation ofLPS with anti-LPS antibody (0.1 μg/ml) decreases LDH release induced byLPS(S) at 6 hours and does not affect LPS(E) toxicity or LPS(S) effects18 hours following addition (FIGS. 4 A and 4B). Thus, cell injuryinduced by LPS from either E. coli or S. enterica can be mitigated byrASS presence in growth media even more consistently than by anti-LPSantibodies. The protective action of rASS is significant at both timepoints studied (6 and 18 hours).

Example 6 LDH Release and MTS Mitochondrial Function Assays

CytoTox 96® Cytotoxicity Assay (Promega, Madison, Wis.) is used toquantitatively measure lactate dehydrogenase (LDH) release to assess therelative number of lysed cells according to the manufacturer'sinstructions. CellTiter 96 AQ Assay (Promega) is used to quantitativelymeasure the conversion of a tetrazolium compound, MTS, into a formazanproduct by the mitochondria of living cells to assess the relative cellviability according to the manufacturer's instructions.

Cytotoxicity and cell death in many tissues involve caspase-3 andcalpain-2 activation that results in a cleavage of several commonproteins such as major cytoskeletal αII-spectrin. In RAW264.7 cells,both LPS types at 1 μg/ml induce all-spectrin breakdown and generationof a 150 kDa fragment (SBDP150) within 18 hours after treatment (FIG.5A), while pretreatment with rASS (1 μg/ml) significantly attenuatesLPS-induced SBDP150 formation. The LDH release and all-spectrinbreakdown in response to LPS are accompanied by a decrease in cellviability as indicated by a mitochondrial respiration activity ofcultured macrophages measured using an MTS reduction assay.Pre-incubation with rASS protects the cells and increases cell viabilityat 18 hours following treatment with LPS (FIG. 5B). In contrast,LPS-induced cell damage assessed by MTS test is not affected by anti-LPSantibody (0.1 μg/ml) (FIG. 5B). Complete blocking of mitochondrialdysfunction caused by both LPS (E) and LPS(S) is achieved at therASS:LPS mass ratio of about 1:1. Finally, rASS is capable of mitigatingendotoxin-induced cellular damage when added after LPS. As shown in FIG.6, addition of rASS 1 hour after LPS nearly abolishes LPS-inducedcytotoxicity in a rASS dose-dependent fashion in mouse macrophages.

Example 7 ELISA Analyses

ASS SW ELISA Assay. The levels of endogenous ASS in serum is determinedby SW ELISA assay (Banyan Biomarkers, Inc.) using polyclonal ASSantibody as capture and mouse monoclonal as detection antibody. Colordevelopment is accomplished using anti-mouse HRP— conjugated Absfollowed by TMB substrate incubation. ASS levels are calculated from acalibration curve using human rASS or PEGylated-rASS prepared as aboveas an ASS standard.

Quantitative detection of TNF-α and CRP in animal blood serum wasperformed, using sandwich ELISA kits from eBioscience Inc. and BioVendorLLC respectively, according to the manufacturer's instructions.

Example 8 LPS and ASS Complex Formation

The rASS preparations are examined for LPS binding affinity by gel-shiftassay of rASS-LPS complex formation. Purified LPS (E. coli), LPS (S.enterica), and rASS alone or in combination are incubated for 1 hour at37° C., then subjected to non-denaturing gel electrophoresis, run with192 mM Tris, 325 mM glycine, (pH 8.3) at 25 mA for 2.5 hours using 4-20%gradient polyacrylamide (PAA) gels (Invitrogen, Carlsbad, Calif.) in atray filled with ice-water slurry. Gels are first pre-run with samebuffer for 15 min. rASS is visualized in the gel by Coomassie Blue as asingle band, while LPS alone is not detected (FIG. 2D). In contrast,this technique reveals the pre-incubated rASS-LPS complexes asheterogeneous patches with a dramatic mobility shift compared to rASSalone (FIG. 2D) and similar to LPS visualized by silver staining.

Example 9 Beneficial Effects of rASS in Rodent Models of Endotoxic Shock

A model of experimental endotoxemia in Balb/c mice is used tocharacterize the efficacy of rASS in protecting an immunologicallyintact host against serious LPS attack. Groups of 6 mice receiveintra-peritoneal (i.p.) injections of saline with either 15 mg/kg E.coli LPS or 15 mg/kg S. enterica LPS followed in 1 hr by infusion with 5mg/kg rASS. Survival is recorded during a period of 32 hr. As shown inFIG. 7A, LPS mice have 80% mortality at 24 hr and all die by 30 hr. Incontrast, 50% mice challenged by LPS and subsequently treated once withrASS at 3:1 mass ratio survive at 32 hr (FIG. 7A).

For survival experiments, Sprague-Dawley rats are randomly divided intothree groups (at least 4 in each group). Group 1 receives an i.p.injection 5 mg/kg rASS, group 2 is given 25 mg/kg E. coli LPS followedin 1 hr by infusion with 5 mg/kg rASS, and group 3 is given 25 mg/kg E.coli LPS alone. Survival is recorded during a period of 72 hr.

In rat endotoxemia model, a bolus injection of LPS (25 mg/kg, i.p.)results in a 72-hr survival of 60% (FIG. 7B). In contrast, 100% ratssurvive when rASS is injected 1 hour following LPS challenge at rASS/LPSmass ratio of 1:5 (FIG. 7B).

Example 10 Effects of rASS on the Release of Endotoxic Shock-RelatedMarkers

To assess endotoxin-induced systemic inflammation and organ injury,several common serum markers are assessed. For these biomarker releasestudies, rats are given i.p. either E. coli LPS (25 mg/kg or 5 mg/kg)alone, or LPS preincubated (1 hr, 37° C.) with rASS (5 mg/kg), or LPSfollowed in 1 hr by infusion with rASS. TNF-α, LDH and CRP levels areassessed in sera prepared from rat blood samples 3 hr (TNF-α detection)or 72 hr (LDH and CRP detection) after LPS injection.

Example 11 Suppression of TNF-α Release and C-Reactive Protein (CRP)Levels by rASS in Rat Endotoxemia

To determine whether the protective activity of ASS is associated withpro-inflammatory cytokine attenuation in vivo we measure the serum TNF-αlevel in endotoxemic rats challenged as in Example 10. As shown in FIG.8A, LPS injected at 25 mg/kg induces TNF-α rise in serum up to about 125pg/ml at 3 hours post injection, while administration of 5 mg/kg of rASSone hour after LPS decreases TNF-α production by 40% (FIG. 8A).Moreover, lower magnitude increases of TNF-α induced by i.p. treatmentwith 5 mg/kg LPS are abrogated by a subsequent injection of 5 mg/kg ofrASS (FIG. 8A).

Concomitant increase in serum CRP levels are abolished by rASS injected1 hr following sub-lethal 25 mg/kg LPS challenge thus returning CRP tocontrol levels (FIG. 8B).

Example 12 Inhibition of Serum LDH Release in Endotoxemia Rats

Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many bodytissues, including the liver. Thus, elevated serum levels of LDHindicate a leakage of LDH from tissues, potentially due to a multi-organdamage elicited by various insults. FIG. 9 shows that LPS administeredto rats at the sub-lethal dose of 25 mg/kg induces nearly 7-foldincrease of LDH in serum compared with control. However, when the LPSchallenge is accompanied or followed by i.p. injection of 5 mg/kg ofrASS, the LDH release is reduced by correspondingly 45% and 49%. (FIG.9)

Example 13 ASS PEGylation Increases rASS Enzymatic Activity

rASS is either used alone or is PEGylated as described in Example 2. APEGylated-rASS and rASS enzymatic activity test is developed accordingto the principle enzymatic reaction catalyzed by ASS followed bydetermination of inorganic phosphate produced by pyrophosphatase (Schema1).

Briefly, 5 μl of sample is added to 45 μl of 10 mM Tris-HCl (pH 7.5)containing 6 mM MgCl₂, 20 mM KCl, 1 mM ATP, and 1 U/ml ofpyrophosphatase plus or minus substrates of 1 mM aspartic acid and 12.5mM citrulline in a 96-well microtiter plate. Inorganic phosphate (Pi)generated in the reaction is determined using malachite green assay kitusing the manufacturer's instructions (RND Systems, Minneapolis, Minn.).

FIG. 10 illustrates greater than 2.5-fold increased in vitro enzymaticactivity of PEGylated rASS relative to rASS alone (p<0.01) unexpectedlyindicating that PEGylation improves the in vitro enzymatic activity ofrASS.

Example 14 ASS PEGylation Increases rASS In Vivo Stability

The stability of PEGylated-rASS is studied and compared to that of rASSalone. 5 mg/kg rASS and PEGylated rASS are injected intraperitoneal(i.p.) in adult male Sprague-Dawley rats (200-225 g) (HarlanLaboratories Inc., Indianapolis, Ind.) housed under constant temperature(22° C.) and humidity with 12 hours light/dark cycle with access to chowand water ad libitum. Blood is collected from heart of anesthetizedanimals 3 h and 20 h after injection, using at least 3 rats for eachtime point. The level of rASS or PEGylated-rASS is determined bySW-ELISA as described in Example 7.

As illustrated in FIG. 11, PEG-rASS is significantly more stable, and isretained in circulation for a much longer time after i.p. injection vs.non-PEGylated rASS. These results combined with the increased enzymaticactivity of PEGylated-rASS indicated that PEGylation produces a morerobust rASS molecule with overall unexpectedly greater ability to bindand clear LPS from a pathogenic Gram-negative bacteria infected subject.

Statistics

Statistical analyses are performed using GraphPad Prism 5 software. Dataare evaluated by 2-tailed unpaired t-test. The criterion for statisticalsignificance is set at p<0.05 or p<0.01.

Sequences

SEQ ID No. 1:  Homo sapiens argininosuccinate synthetase 1 (ASS) protein (412 aa): (SEQ ID NO: 1)MSSKGSVVLAYSGGLDTSCILVWLKEQGYDVIAYLANIGQKEDFEEARKKALKLGAKKVFIEDVSREFVEEFIWPAIQSSALYEDRYLLGTSLARPCIARKQVEIAQREGAKYVSHGATGKGNDQVRFELSCYSLAPQIKVIAPWRMPEFYNRFKGRNDLMEYAKQHGIPIPVTPKNPWSMDENLMHISYEAGILENPKNQAPPGLYTKTQDPAKAPNTPDILEIEFKKGVPVKVTNVKDGTTHQTSLELFMYLNEVAGKHGVGRIDIVENRFIGMKSRGIYETPAGTILYHAHLDIEAFTMDREVRKIKQGLGLKFAELVYTGFWHSPECEFVRHCIAKSQERVEGKVQVSVLKGQVYILGRESPLSLYNEELVSMNVQGDYEPTDATGFININSLRLK EYHRLQSKVTAKSEQ ID No. 2:  Homo sapiens argininosuccinate synthetase 1 (ASS) cDNA (1239 nt): (SEQ ID NO: 2)ATGTCCAGCAAAGGCTCCGTGGTTCTGGCCTACAGTGGCGGCCTGGACACCTCGTGCATCCTCGTGTGGCTGAAGGAACAAGGCTATGACGTCATTGCCTATCTGGCCAACATTGGCCAGAAGGAAGACTTCGAGGAAGCCAGGAAGAAGGCACTGAAGCTTGGGGCCAAAAAGGTGTTCATTGAGGATGTCAGCAGGGAGTTTGTGGAGGAGTTCATCTGGCCGGCCATCCAGTCCAGCGCACTGTATGAGGACCGCTACCTCCTGGGCACCTCTCTTGCCAGGCCCTGCATCGCCCGCAAACAAGTGGAAATCGCCCAGCGGGAGGGGGCCAAGTATGTGTCCCACGGCGCCACAGGAAAGGGGAACGATCAGGTCCGGTTTGAGCTCAGCTGCTACTCACTGGCCCCCCAGATAAAGGTCATTGCTCCCTGGAGGATGCCTGAATTCTACAACCGGTTCAAGGGCCGCAATGACCTGATGGAGTACGCAAAGCAACACGGGATTCCCATCCCGGTCACTCCCAAGAACCCGTGGAGCATGGATGAGAACCTCATGCACATCAGCTACGAGGCTGGAATCCTGGAGAACCCCAAGAACCAAGCGCCTCCAGGTCTCTACACGAAGACCCAGGACCCAGCCAAAGCCCCCAACACCCCTGACATTCTCGAGATCGAGTTCAAAAAAGGGGTCCCTGTGAAGGTGACCAACGTCAAGGATGGCACCACCCACCAGACCTCCTTGGAGCTCTTCATGTACCTGAACGAAGTCGCGGGCAAGCATGGCGTGGGCCGTATTGACATCGTGGAGAACCGCTTCATTGGAATGAAGTCCCGAGGTATCTACGAGACCCCAGCAGGCACCATCCTTTACCATGCTCATTTAGACATCGAGGCCTTCACCATGGACCGGGAAGTGCGCAAAATCAAACAAGGCCTGGGCTTGAAATTTGCTGAGCTGGTGTATACCGGTTTCTGGCACAGCCCTGAGTGTGAATTTGTCCGCCACTGCATCGCCAAGTCCCAGGAGCGAGTGGAAGGGAAAGTGCAGGTGTCCGTCCTCAAGGGCCAGGTGTACATCCTCGGCCGGGAGTCCCCACTGTCTCTCTACAATGAGGAGCTGGTGAGCATGAACGTGCAGGGTGATTATGAGCCAACTGATGCCACCGGGTTCATCAACATCAATTCCCTCAGGCTGAAGGAATATCATCGTCTCCAGAGCAAGGTCACTGCCAAATAG

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified. Methods of nucleotide amplification,cell transfection, and protein expression and purification are withinthe level of skill in the art.

Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference for the material for which each reference iscited as well all other material taught therein.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

The invention claimed is:
 1. A process of treating exposure of a subjectto a bacterial endotoxin, comprising: administering a therapeuticallyeffective amount of argininosuccinate synthetase to a subject exposed toa bacterial endotoxin.
 2. The process of claim 1 wherein saidargininosuccinate synthetase is recombinantly expressed.
 3. The processof claim 1 wherein said argininosuccinate synthetase is PEGylated. 4.The process of claim 1 wherein said argininosuccinate synthetase isPEGylated with a PEG comprising PEG₁₂.
 5. The process of claim 1 whereinsaid argininosuccinate synthetase is PEGylated with an unbranched PEG, abranched PEG, or combinations thereof.
 6. The process of claim 5 whereinsaid argininosuccinate synthetase and said PEG are present in a ratioranging from 1:1 to 1:400.
 7. The process of claim 1 wherein saidsubject is infected with a pathogenic Gram-negative bacteria.
 8. Anisolated protein preparation for performing the process of claim 1comprising: a PEGylated argininosuccinate synthetase protein.
 9. Thepreparation of claim 8 wherein said argininosuccinate synthetase israndomly PEGylated.
 10. The preparation of claim 8 wherein saidargininosuccinate synthetase is site-directed PEGylated.
 11. Thepreparation of claim 10 wherein said argininosuccinate synthetase issite-directed PEGylated at one or more cysteines, lysines, histidines,or combinations thereof.
 12. The preparation of claim 8 wherein saidPEGylation comprises PEG₁₂ covalently associated with saidargininosuccinate synthetase by an amide bond.
 13. The preparation ofclaim 12 wherein said PEG ₁₂ is a component of a branched PEG.
 14. Thepreparation of claim 8 wherein said PEGylation comprises branched PEG.